Our studies have led to the identification of a novel growth regulatory pathway in non-small cell lung cancer (NSCLC) that involves CYP24, the enzyme that degrades the endogenous anti-proliferative agent, 1,25- dihydroxyvitamin D3 (1,25(OH)2D3). The level and biological activity of 1,25(OH)2D3 in tissues is normally controlled by maintaining a precise balance between the rates of its synthesis by CYP27B1 and degradation by CYP24. We discovered that this balance is dysregulated in NSCLC. Specifically, we determined that CYP24 is overexpressed in human NSCLC cells and a majority of human lung tumors. We hypothesize that CYP24 overexpression facilitates lung cancer growth by allowing neoplastic cells to escape the anti- proliferative effects of locally-produced 1,25(OH)2D3. Accordingly, we found that CYP24 inhibition uncovers 1,25(OH)2D3 production by CYP27B1+CYP24+ NSCLC cells and allows for accumulation of hormone in amounts sufficient for growth suppression. Our data further indicate that Ets-1 and C/EBP proteins increase CYP24 transcription in NSCLC cells. Activation of these transcription factors has previously been linked to oncogenic signaling in lung cancer. Our objective is to establish the effect of aberrant CYP24 expression on lung cancer growth and the mechanism for CYP24 overexpression in lung cancer. In Aim 1 we will test the prediction that CYP24 overexpression facilitates NSCLC growth by decreasing the stability and anti- tumor activity of 1,25(OH)2D3. We will quantify the effect of modulating CYP24 expression on 1,25(OH)2D3 catabolism and anti-tumor activity in NSCLC cell lines and tumor xenograft models. We will also use an innovative ex vivo assay to determine the association between CYP24 expression and 1,25(OH)2D3 catabolism in human lung tumors. In Aim 2 we will test the prediction that CYP24 overexpression antagonizes autocrine growth regulation by vitamin D3 in NSCLC. We will utilize a novel CYP24 inhibitor to demonstrate the effect of CYP24 on 1,25(OH)2D3 synthesis by CYP27B1+CYP24+ NSCLC cells and demonstrate the ability of locally- produced hormone to elicit biological responses. We will also test whether CYP24 overexpression and CYP27B1 loss are independently correlated with the lung cancer phenotype. In Aim 3 we will test the prediction that oncogene-activated Ets-1 and C/EBP transcription factors regulate CYP24 expression in NSCLC. The effect of Ets-1 and C/EBP modulation on CYP24 transcription and activity will be established. Recruitment of Ets and C/EBP proteins to the endogenous CYP24 promoter (as a function of ras activation) will be evaluated by chromatin immunoprecipitation. The proposed studies will identify a novel mechanism for the pathogenesis of NSCLC in which oncogene-activated transcription factors facilitate tumorigenesis by increasing the expression of a protein (CYP24) that catabolizes an endogenous growth suppressor (1,25(OH)2D3). The therapeutic implication of this paradigm, once confirmed, is that agents that inhibit CYP24 can be used to restore vitamin D3-mediated growth control in NSCLC.